Production of metallothionein polyclonal antibodies using chickens as model.
The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.
Ortiz-Bueno
Angélica María
AM
Laboratorio de Investigaciones Químico Clínicas, Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, México.
León-Chávez
Bertha Alicia
BA
Ruiz-Tagle
Alejandro
A
Lozano-Zarain
Patricia
P
Castro-Caballero
Leopoldo
L
Achanzar
William E
WE
Brambila
Eduardo
E
D000818:Animals; D002645:Chickens / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D007136:Immunoglobulins / Q000096:biosynthesis* / Q000276:immunology; D008668:Metallothionein / Q000276:immunology* / Q000302:isolation & purification; D020033:Protein Isoforms / Q000276:immunology / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar
C016512:IgY; D007136:Immunoglobulins; D020033:Protein Isoforms; D008668:Metallothionein